Coating of Platelets with Antibody in vitro

OUTLINE


Antibody-coated platelets (opsnized) may be used in the subsequent thrombophagocytosis assay.

1. Resuspend 1.6x10^8 of CMFDA-labeled platelets in 200µl of BSGC-BSA (v/w 1%) either with a control Ab or an Ab directed to platelet Ag (you may use Grener F-bottom microtitration plate for this step). If the Ab used is rat IgG1 (k) against mouse CD41 (MWReg30, Becton Dickinson, Cat.: 09911D) then the advised concentration is 0.6µg/ml (0.15µg per 200µl of suspension). Incubate the suspension for 45 min., at 37ѓC.
2. Centrifuge at 3000 rpm at 4ѓC, for 7 min.
3. Resuspend in BSGC.
4. Centrifuge at 3000 rpm at 4ѓC, for 7 min.
5. Resuspend in 200µl of IMDM.
6. Centrifuge at 3000 rpm at 4ѓC, for 7 min.
7. Resuspend in 200µl of IMDM-FCS 5% decomplemented.
   

1. BSGC
   
2. BSGC-BSA 1% (v/w) = 9990ml BSGC+10g BSA
3. IMDM (Iscove's Modified Dulbecco's Medium)
   
4. IMDM-FCS 5% (v/v) heat-decomplemented (56ѓC, 30min).